组蛋白修饰H3K4me3在增强子元件上的选择性标记和功能研究

Enhancers are an important class of regulatory elements and are widely studied in recent years. They can positively regulate the transcription of downstream genes, and play a vital role in the establishment of tissue-specific transcriptional programs in higher organisms. In general, active enhancers are marked by several related histone modifications including H3K4me1 and H3K27ac, while H3K4me3 modification typically appears at gene promoters. Several recent studies reported that in some special conditions H3K4me3 can also appears on enhancers, but it’s still unclear whether H3K4me3 is naturally linked with the functional activity of enhancers. Based on integrative analysis of a large set of omics data and computational modeling, we found in many blood cell lineages a large fraction of active enhancers carry considerable H3K4me3 mark, and these enhancers generally have different transcription factor binding pattern and enhancer RNA transcription level compared to the other active enhancers. Based on these findings, we plan to systematically study the functional difference between the enhancers marked and not marked by H3K4me3, and quantitatively model the correlation between the level of H3K4me3 mark and that of other related epigenetic marks at enhancers as well as the functional activity of enhancers, detect the epigenetic factors regulating enhancer H3K4me3 levels in blood cell lineages, and dissect the functional impact of the selective appearance of H3K4me3 mark on many enhancers in these cells as well as the underlying molecular mechanism, which can greatly help to improve our knowledge of the epigenetic landscape of enhancer elements as well as how they carry out their functions.

增强子是一类广受关注的调控元件，对于组织特异性基因表达谱的建立至关重要。一般认为活性增强子携带H3K4me1等组蛋白修饰，而H3K4me3修饰主要出现在基因启动子。近年一些研究报道了特殊情况下H3K4me3修饰在增强子上的出现，说明对该修饰与增强子之间的联系仍急需进一步全面系统的研究。申请人基于海量组学数据的整合分析和计算建模，发现许多血液和淋巴细胞中大量增强子被H3K4me3标记，并且它们相对其它增强子具有不同的转录因子组合和增强子RNA转录水平等特征。在此基础上，本项目拟系统比较H3K4me3标记的活性增强子与其它增强子之间的差异，定量刻画该修饰与增强子上其它表观修饰以及增强子活性之间的关联，发掘在血液和淋巴细胞中调控增强子上H3K4me3修饰水平的表观因子，深入解析该修饰在血液和淋巴细胞中选择性大范围标记活性增强子的功能影响和分子机制，进一步完善对于增强子表观图象的认识。

人类基因组中存在数以万计的增强子元件。它们通过远距离互作调控基因的组织特异性表达，控制了人类组织的发育和病变。一般认为H3K4me1和H3K9/27ac等组蛋白修饰标记激活状态的增强子，而H3K4me3修饰标记转录激活基因的启动子。一些最新研究发现在特定基因如RAC7被敲除的情况下，H3K4me3修饰会广泛出现在增强子上，并使之进入“超激活”状态。但是，天然状态的细胞中是否存在这种“超激活”增强子未被系统研究过。我们前期通过分析人类不同细胞类型的海量表观组、转录组等多维组学数据，发现在很多血液和淋巴细胞中，存在大量活性增强子携带可观水平的H3K4me3修饰，并将其命名为H3K4me3阳性增强子。项目执行中，我们经过探索和优化，建立了H3K4me1后接H3K4me3的双标记reChIP-qPCR/seq实验体系，验证了这些增强子上两种修饰的染色质共定位，并指出这是它们区别于活性基因启动子和其它H3K4me3阴性活性增强子的主要特征。在此基础上，在K562白血病细胞和GM12878淋巴母细胞中系统地定义了H3K4me3阳性和阴性两类活性增强子，并进行广泛深入的功能对比分析，发现H3K4me3阳性增强子在组蛋白乙酰化、主调控因子结合、增强子RNA转录、超级增强子出现频率等增强子功能活性相关信号上明显高于H3K4me3阴性增强子，并发现这些增强子的下游基因更加富集与红细胞和淋巴细胞分化相关通路，表明H3K4me3修饰在活性增强子上的选择性添加具有重要生物学意义。最后，我们系统利用基于CRISPR技术的基因敲除、ChIP-seq、RNA-seq等实验和数据分析，揭示了KDM5C等表观因子在K562白血病细胞中直接调控了H3K4me3阳性增强子上的H3K4me3修饰水平，并发现KDM5C敲除导致细胞周期基因表达失调和细胞增殖变慢，为进一步研究这些增强子在白血病发展中的作用指明了方向。

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